The existence of two systems of vasoactive polypeptides, a hypertensor and a hypotensor, in mammal organism is quite new. The fundamental bases for understanding the hypertensor system, renin-angiotensin system were established through papers of Houssay and Fasciolo (1937), Houssay and Taquini (1938), Braun-Menendez, Fasciolo, Leloir and Munoz (1939) and Kohlstaedt, Helmer and Page (1938). On the other hand, the hypotensor system, kallikrein-kinin system are based on Frey, Kraut and Werle papers, carried out in the 1930 decade (Frey, Kraut and Schultz, 1930; Kraut et al, 1930; Werle, 1936; Werle et al, 1937) as well as Rocha and Silva, Beraldo and Rosenfeld (1949) and Prado, Beraldo and Rocha e Silva (1950).
In the two systems the vasoactive peptide is released to its plasmatic protein precursor through limited proteolysis according to the following general scheme:

Several papers on purification and characterization of proteases and substrates involved in these two systems allowed the clarification of several steps necessary for releasing the active peptide. However, the physiological role of the latter, as well as its catabolism is not totally clarified yet.
The Renin-Angiotensin System
Renin is an acid protease (E.C. 3.4.99.19), produced and stored by juxtaglomerular cells from afferent arteriole of the renal glomerulus (Kohlstaedt et al, 1938; Hartroft, 1963 and Tobian, 1960). The subtract under which this enzyme acts is a plasmatic α2-globulin, angiotensinogen, from which part of the N-terminal sequence is known (Braun-Menendez et al, 1939; Bumpus et al, 1958; Schwyzer and Turrian, 1960) which corresponds to: Asp3-Arg2-Val3 -Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Leu11 -Val12-Tyr13-Ser14.
When renin hydrolisates the Leu10-Leu11 bond in the angiotensinogen molecule, decapeptide angiotensin I, which is as not very potent vasoconstrictor, is released. A second enzyme, described by Skeggs et al (1956), called converting enzyme, is the responsible by the hydrolysis of the Phe8-His9 bond and by releasing octapeptide angiotensin II, which is pharmacologically active, being inactivated by angiotensinases.
The Kallikrein-Kinin System
The kallikrein-kinin system comprises kininogenases which hydrolisates an inactive precursor, the kininogen, and releases kinins, which are inactivated by kininases.
The expression kininogenase comprises proteases, such as: kallikreins, trypsin, pepsin, some bacterian proteases and snake poison (Prado, 1970; Rocha e Silva et al, 1949; Suzuki and Iwanaga, 1970). Among these enzymes, kallikreins are specifics for the system: these are serine—proteases that release kinins of the kininogen, by limited proteolysis (Neurath, 1975) and have low proteolitic activity on other proteins. Two types of kallikreins are found in mammals: glandular and plasmatic, which are different each other concerning to physical-chemical and immunological proprieties, reaction velocity with kininogen and synthetic subtracts, types of kinins released and responds to a great variety of synthetic and natural inhibitors.
On the other hand, kininogens are acid glycoproteins which contains a bradykinin molecule in the C-terminal or next to it (Pierce, 1968); they are hydrolisated by glandular kallikreins, releasing lysyl-bradykinin, as well as by plasmatic releasing bradykinin (Rocha e Silva, 1974). Lysyl-bradykinin is converted to bradykinin by the existing aminopeptidases contained in plasma (Erdös and Yang, 1970) as well in tissues (Hopsu et al, 1966 a, b; Borges et al, 1974; Prado et al, 1975).
Two kininogens functionally different have been described in plasma, namely, a high molecular weight kininogen, which is subtract for the two kallikrein (plasmatic and glandular), and a low molecular weight, which is a good subtract only for glandular kallikrein (Werle and Trautschold, 1963; Prado et al, 1971).
Bradykinin (BK=Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), lysyl-bradykinin and methionyl-lysyl-bradykinin are strong physio-pharmaco-pathological agents, which produces hypotension and vasodilation, pain, contraction of the smooth muscle, increases vascular permeability and leukocyte migration (Erdös and Yang, 1970; Pisano, 1975).
Physiological Role of Kinins:
The action of kinins in the organism is not totally clarified, although some attributions have been indicated them as participating in several physiological functions, either at systemic or tissular levels.
It is proposed its mediation in different processes such as: peripheric vasodilatation and mediation in inflammatory phenomena; interaction with the synthesis system and prostaglandins release; mobility of spermatozoids; renal flux regulation; mediation in the sodium reabsorption by nephron (Wilhelm, 1973; Terragno et al, 1975; Baumgarten et al, 1970; Schill and Haberland, 1974; Levinsky, 1979).
In order to clarify the exact role carried out by kinins in this process, it is important to know not only the mechanism that leads to its release but also to its catabolism.
Catabolism of Kinins:
The responsible enzymes for inactivation of kinins are generically known as kininases. Under this acronym it is comprised a series of peptidases which are capable to hydrolysate the bonds in BK molecule or its derivatives, not being necessary or prove to be participant of the kinins catabolism.
Observations on the existence of such class of enzymes have been carried out since the initial researches of the kinins system and have been described in several organs, tissues and physiological liquids by many researchers groups.
Plasma
Two types of kininases have been characterized already in the human plasma: kininase I (arginine carboxypeptidase, E.C. 3.4.12.7) and kininase II (peptidyl-dipeptidase, E.C. 3.4.15.1).
Kininase I is a carboxypeptidase type enzyme, which was purified for the first time from Cohn fraction IV (Erdös e Sloane, 1962). This enzyme hydrolysates the Phe8-Arg9 bonds of BK. It was originally called carboxypeptidase-N because of its proprieties, which make them different of pancreatic carboxypeptidase B. Although its official name, arginine carboxypeptidase, this enzyme catalyses better the lysine C-terminal hydrolysis than arginine, in many substrates (Oshima et al, 1975).
Among synthetic substrates used in the purification and specificity studies of such enzyme it can be cited the HLA (Oshima et al, 1975).
The second enzyme having kinin activity, described in plasma, is the kininase II, which inactivate BK by hydrolysis of the Pro7-Phe8 bonds and releases Phe-Arg dipeptides (Yang and Erdös, 1967).
Later, it was observed that such enzyme is identical to the angiotensin I converting enzyme (AI) of the renin-angiotensin system (Yang et al, 1970a and Yang et al, 1970b), therefore, being, responsible by hydrolysis of the Phe8-His9 bond of the AI molecule. One of the features of such enzyme is that it is inhibited by potentiator peptides of BK (BPP), described by Ferreira (1965), and Ferreira (1966).
It was also described in other animal species enzymes with similar specificity to those kininases I e II from human plasma (Erdös and Yang, 1970).
Lung
Great importance has been attributed to lung in which concerns BK elimination; many papers published by the literature describes the inactivation by this organ, regarding the high percentage of BK infused (Ferreira and Vane, 1967; Biron, 1968 and Dorer et al, 1974).
The kininase II has been already purified in hog lung (Dorer et al, 1972 and Nakajima et al, 1973) rabbit lung and rat lung (Soffer et al, 1974 and Lanzillo and Fanburg, 1974).
Studies of Ryan et al have contributed to clarify the mechanism of inactivation of this organ. BK would be inactivated, while AI would be converted into AII during the circulation, by an enzyme kininase II type that was in the pynocitotic vesicles of the vascular endothelium. Ryan et al, also observed that BK is much more easily hydrolyzed than LBK-BK and MLBK. Their theory is that these bigger kinins have a more difficult access to the vesicles. According to Ryan et al statement the BK hydrolysis products which were found after the lung circulation would be consequence not only from the action of the first cited enzyme but also from the action of other enzymes contained in the cytoplasm of endothelial cells (Ryan et al, 1968 and Ryan et al, 1975).
Liver:
Erdos and Yang attributed almost exclusively to the plasmatic and pulmonary kininases the responsibility for the kinin catabolism “in vivo”. Researches carried out by Prado et al (1975) show, however, that other organs are able to inactivate kinins when they are perfused in weak rats, in which lung circulation was excluded from the perfusion circuit. In the referred paper when liver is perfused “in situ”, it was shown that the organ inactivates considerable quantity of BK.
Following these researches, Borges et al (1976) observed that BK inactivation by the perfused liver “in situ” is due to, at least, two enzymes: a peptidyl dipeptide hydrolase and a second one that hydrolyzes the Phe5-Ser6 bond of BK. This enzyme could be a membrane peptidase, since it was removed from the perfused liver through the use of Triton X-100 in the perfusion liquid. According to the authors, the kinin activity obtained in this research is, very low when compared to those found in the supernatant of the total homogenate of the organ.
Mazzacoratti (1978) have worked with a preparation of this type, that is, homogenized liver from rats. It was purified two serine-proteases having different molecular weight which hydrolisates the Phe5-Ser6 bond of BK.
Brain
It has been studied by many researchers the metabolism of kinins in brain extracts (Iwata et al, 1969; Camargo et al, 1969).
Kininases from homogenized rabbit brain have been systematically studied by Camargo et al (1973), Oliveira et al (1976). Two thiol-endopeptidases optimum pH 7.5 were purified from the supernatant fraction. The first enzyme, kininase A, hydrolyzes the Phe5-Ser6 BK bond and has a molecular weight of 71 kDa; while the other, kininase B, hydrolysates Pro7-Phe8 as kininase II, but it has a molecular weight of 6900. This enzyme would be different from the converting enzyme (kininase II), since preliminary studies did not show the conversion of AI into AII.
Wilk, Pierce and Orlowiski (1979) described two enzymes from brain tissue which differs from the referred above. One of the enzymes, which was extracted from the bovine pituitary, also hydrolysates the Phe5-Ser6 Bk bond, however because of its molecular weight (higher than 100000) and because it is inhibited by Na+ and K+ it differs from kininase A. The second enzyme described, which was extracted from rabbit brain, is specifically for hydrolysis of those peptide bonds in which proline contributes with carboxyl group. This enzyme firstly hydrolyses the Pro7-Phe8 BK bond and secondly, the Pro3-Gly4 bond.
Kidney:
The kininase activity of kidney is higher than found in plasma or liver (Erdös and Yang, 1970).
Several enzymes have been purified, in this organ, with kininase activities. Researches of Erdos et al, have identified three different enzymes in the kidney: one, carboxypeptidase type, which releases arginine C-terminal of BK, which differs from some properties of plasmatic kininase I, this is why it was called kininase p (Erdös and Yang, 1966); another enzyme, which hydrolisates the Pro7-Phe8 bond (Erdös and Yang, 1967), and a third one, characterized as an imidopeptidase, which inactivates BK by hydrolysis of the Arg1-Pro2 bond (Erdös e Yang, 1966).
Koida and Walter (1976) purified, from sheep kidney, an enzyme that hydrolysates Pro-x bonds type in the molecule of several peptides, among which the BK. It was observed that the x aminoacid cannot be proline and that its catalysis is faster if x is a lipophilic aminoacid.
The kinin catabolism by the kidney has been studied by methods aiming at to identify the inactivation sites of such peptides. These studies indicate that, besides the BK hydrolysis that occurs at vascular network level, the catabolism of kinins by enzymes located at renal cortical cells seems to have great importance (Erdös and Yang, 1967).
The kininase activity is very low in the glomerulus, but a type II kininase is found in great concentration in brush border of the proximal convoluted tubule (Holl et al, 1976, Casarini et al, 1997). In agreement with this discovery, Oparil et al (1976) observed that a high percentage of BK microinfused is inactivated in the proximal tubule. Considering that the kinin generation in kidneys should occur close to the distal tubule, where kallikrein is synthesized (Ørstavik et al, 1976), it seems to be logical to suppose that from this point, other kininases should be present and the nephron or in the intratubular fluid.
Urine
A carboxypeptidase was well characterized by Erdös et al (1978), in human urine; it releases BK C-terminal arginine and differs from the plasmatic one as to molecular weight, inhibitors action and immunological proprieties. However, kinetic and inhibition similarities to renal enzyme are shown.
Ryan et al described, in 1978, three enzymes contained in urine: one enzyme hydrolyzates the Pro7-Phe8 BK bond and transform AI into AII; another enzyme, having 63 kDa molecular weight, which breaks the Phe8-Arg9 BK bond, is not inhibited by BPP9a.
Figueiredo et al (1978) also described, a kininase having molecular weight of 250 kDa, which is inhibited by chelate agents that would be similar to the third among those described by Ryan. This enzyme hydrolysates C-terminal arginine, although it does nor hydrolysates the HLA synthetic substrate.
With the exception of Erdös et al, 1978, research that have purified and characterized a carboxypeptidase from urine, all researches, however, the described enzymes were only partially purified and/or characterized. Due to these contradictory data, the present invention aims at to characterize the different kininase activities that are the ACE in human urine.
One of the forms of low molecular weight (LMW) of the angiotensin I converting enzyme of 91 kDa was observed during the preparation of such enzyme from rat lung homogenate [Lanzillo et al, 1977]. This LMW form from ACE was also observed in human lung [Nishimura et al, 1978], hog kidney [Nagamatsu et al 1980] and human kidney [Takada et al, 1981]. Iwata et al (1983) and Yotsumoto et al (1983) have shown that ACE LMW of 86-90 kDa can be obtained from rabbit lung and human plasma, respectively, after treatment with bases. In the 90′ Lantz et al (1991), described three different ACE isoforms having molecular weights of 150 kDa, 80 kDa and 40 kDa characterized in the human cerebrospinal fluid. All the previously referred are similar to somatics. Casarini et al, 1991, 1995, 2001, described the 65 kDa and 90 kDa isoforms, both N-domain in hypertense patients urine and 65 kDa on normal persons. Deddish et al (1994) purified an ECA with 108 kDa molecular weight in ileal fluid, which is also an N-domain isoform of ACE.
It has been described the purification of several isoforms of ACE [Ryan et al, 1978; Kokubo et al, 1978; Skidgel et al, 1987; Casarini et al, 1983, 1987]. Kokubo et al (1978) found three different forms of ACE normal human urine. Two forms with high molecular weight of >400 kDa and 290 kDa and a third one, molecular weight of 140 kDa. Ryan et al (1978) described a kininase II human urine that was separated in two forms. The first co-cromatography with somatic ACE of 170 kDa, and the second was similar to a protein having molecular weight of 90 kDa. Casarini et al (1983, 1991, 1992, 1995, 2001) described he ACE in human urine of normal persons and hypertense patients with molecular weights of 190 kDa, 90 kDa and 65 kDa and also in rat urine (Casarini et al 1987). Alves et al, 1992 also described isoforms of 170 kDa, 90 kDa and 65 kDa in urine of normal persons and hypertense patients. Costa et al, 1993, 2000 described in normal persons urine, ACE with different molecular weights of 170 kDa, 65 kDa and 59 kDa, and in the hypertense patients, renovasculares enzymes with molecular mass of 55 kDa, 57 kDa e 94 kDa. The ACE activity in urine is not from the plasma but from the renal tubule (Casarini et al, 1997) and can be used as a reference for the renal tubular damage, since there is a considerable level increasing in renal and infections of upper urinary treat diseases [Baggio et al, 1981; Kato et al, 1982].
It was also recently described, two ACE isoforms in intracellular and extracellular medium of mesangial cells in culture, having molecular weight of 130 kDa and 65 kDa (Andrade et al, 1998). It was still observed the presence of 190 kDa and 65 kDa isoforms in children urine but in premature children only 65 kDa isoforms ACE, being the latter similar to the N-domain portion of the same. In premature children, it was found, in a period of 1 to 30 days after they were born, that these 190 kDa isoform would appear only in the thirtieth day (Hattori et al, 2000).